Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis

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Sybille Koehler,1,2 Alexander Kuczkowski,1 Lucas Kuehne,1 Christian Jüngst ,3 Martin Hoehne ,1,3 Florian Grahammer,4 Sean Eddy ,5 Matthias Kretzler ,5,6 Bodo B. Beck,7 Jörg Höhfeld,8 Bernhard Schermer,1,3 Thomas Benzing,1,3 Paul T. Brinkkoetter,1 and Markus M. Rinschen1,3,9

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. Methods We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. Results Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. Conclusions We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype- to-protein correlation.

JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019030312

Received March 28, 2019. Accepted December 4, 2019. Podocytes are specialized epithelial cells at the kid- P.T.B. and M.M.R. shared senior authorship. ney ﬁltration barrier that enwrap the glomerular capillaries.1 Upon injury, podocytes dedifferenti- Published online ahead of print. Publication date available at www.jasn.org. ate, lose their unique three-dimensional morphology, and detach into the urine. This response to Correspondence: Dr. Paul Brinkkoetter, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of injury of any kind is morphologically followed Cologne, Faculty of Medicine and University Hospital Cologne, and accompanied by glomerular scarring and Kerpener Str.62, Köln, Germany 50931, or Dr. Markus Rinschen, Center for Metabolomics and Mass Spectrometry, The Scripps Re- FSGS. Various molecular, chemical, and genetic search Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037. Email: stressors can induce such a response. Diverse animal [email protected] or [email protected] models are used to study the disease. Commonly Copyright © 2020 by the American Society of Nephrology

JASN 31: ccc–ccc,2020 ISSN : 1046-6673/3103-ccc 1 BASIC RESEARCH www.jasn.org used models for identifying cellular pathways during podo- Significance Statement cyte injury include genetic models, and chemically induced podocyte damage such as the Doxorubicin nephrosis and LPS Analyses of entire glomeruli using a proteomic, transcriptomic, or models. Although these models are widely used, it is currently other “omic” approach may obscure the molecular footprints of not clear which parts of human podocyte disease are reflected early and decisive processes in podocytes responding to injury. To pinpoint mechanisms underlying glomerulosclerosis, the authors in the animal models. Even with an increased understanding performed ultrasensitive proteomics of purified podocyte fractions of the genomic landscape of FSGS,2,3 the immediate molec- at early injury stages in mouse models of glomerular disease in- ular response of podocytes in response to injury is still induced by doxorubicin or LPS. These analyses revealed an early completely understood at a systems level. stress response that involves upregulation of metabolic, proteo- fi Although proteomics technology is increasingly used to static, and mechanoresponsive mechanisms. They also identi ed conserved upregulated proteins involved in the podocyte stress 4–7 study glomerular disease, the three-cell architecture of the response, including the mechanosensor Filamin-B, and found a high glomerulus limits data interpretation of glomerular omics correlation between proteinuria and Filamin-B levels. The work data: podocyte injury leads to podocyte loss, and thereby alters demonstrates that proteome integration at the single glomerulus the cellular composition of the glomerulus.8 In addition, the and the individual organism levels can link “omics” datasets to technical nature of proteomics requires analysis of pooled ma- physiological function at high resolution. terial from several animals with variable phenotypes, limiting feasibility of these studies. To improve this, we adapted an ultra- HBSS) and 500 ml Dynabeads in digestion buffer (containing sensitive proteome analysis9,10 of pure podocyte fractions from collagenase 300 U/ml [Collagenase Type II; Worthington], individual mice comprising as few as 10,000 cells to identify 1 mg/ml pronase E [P6911; Sigma, Germany], and DNase I proteins that are regulated in the podocytes’ damage response. 50 U/ml [A3778; Applichem, Germany]). Kidneys were The use of mouse models allows studying of early disease minced into 1-mm3 pieces and incubated in digestion buffer stages, when transcriptomic and proteomic changes are al- at 37°C for 15 minutes. The suspension was mildly pressed ready measurable, but podocyte cell number is not yet dimin- through a 100-mm straining sieve for 15 minutes with enough ished. The technique described herein can therefore be used to HBSS buffer (approximately 20 ml). The suspension was then compare different disease stages in mouse models in order to pelleted by mild centrifugation (3000 rpm, 5 minutes), and the identify genes and proteins involved in the early and late dis- solution was resuspended. For primary podocyte isolation, ease responses of podocytes. glomeruli were resuspended in digestion buffer. For RNA isolation, glomeruli were transferred into Trizol until further processing. For proteomic analysis, glomeruli were digested METHODS until a single-cell suspension was obtained which was further used for FACs sorting. For this purpose, glomeruli were in- Transgenic Mouse Models cubated at 37°C for 40 minutes, and the suspension was mixed The Doxorubicin study was performed with R26mTmG mice, by pipetting up and down every 10 minutes. Magnetic parti- which were mated with hNphs2.PodCre mice to achieve GFP cles were discarded. Purity of cells was checked by fluorescence expression exclusively in podocytes.11,12 For the LPS study analysis. Cell suspension (2 ml) was sieved through a 40-mm Podocin.2A.iCre.2A.mTomato mice were used, which express mesh and washed with 10 ml HBSS. Cells were collected by tomato only in podocytes.13 In the Doxorubicin study we used centrifugation at 1500 rpm for 5 minutes at 4°C, resuspended only male mice, whereas in the LPS study we included mice in 0.5 ml HBSS, and supplemented with 0.1% BSA plus DAPI 1 from both sexes. Animals used in the Doxorubicin study were (1 mg/ml). To separate GFP-expressing (GFP )andGFP- 2 on a pure CD-1 background, whereas mice used in the LPS negative (GFP ) cells, glomerular cells were sorted by FACS study were backcrossed for nine generations from C57BL/6 to for the respective dyes. The minimum number of sorted po- CD-1 (95% CD-1). The mouse holding was done in the Uni- docytes was approximately 12,500 podocytes/animal. versity of Cologne animal facility according to standardized specific pathogen–free conditions. The experimental protocol RNA Isolation and Quantitative PCR was examined and approved by the LANUV NRW (Landesamt Glomeruli used for quantitative PCR were isolated from für Natur, Umwelt und Verbraucherschutz Nordrhein- 12-week-old pure Balb/C mice either treated with Doxorubi- Westfalen, State Agency for Nature, Environment and Consumer cin (12 mg/kg body wt) or without treatment. RNA was iso- Protection North Rhine-Westphalia, AZ 84–02.04.2013.A375). lated using the Direct-Zoll RNA MiniPrep Kit according to manufacturer’s instructions (Zymo, Irvine). A primer pair Isolation of Primary Podocytes specific for murine Filamin-B was used to assess Filamin-B Isolation of primary podocytes was performed after euthaniz- mRNA levels in glomeruli: sense primer: 59-CAAAGCTGG ing mice and glomerular preparation was as previously de- GTCCAACATGC-39, anti-sense primer: 59-CGAGTCAAG scribed.14 A detailed protocol was described before. Mice TCTAGGGCACC-39. For normalization, a primer pair spe- were killed by cervical dislocation and isolated kidneys were cific for the house-keeping gene HPRT was used: sense: 59- manually perfused with Dynabeads suspension (200 ml/10 ml GCTGACCTGCTGGATTACAT-39,anti-sense:59-TTGGGG

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CTGTACTGCTTAAC-39. cDNA synthesis was performed us- Cologne. As control, we used a cancer biopsy specimen with- ing the High Capacity cDNA RT-Kit (Applied Biosystems, out glomerular injury. Immunohistochemistry was per- Foster City). Quantitative PCR was performed using SYBR formed as mentioned above using mouse tissue. Green (Thermo Fisher, Waltham) according to the manufacturer’s instructions. Drosophila melanogaster Strains and Holding Fly strains were obtained from the Vienna Drosophila Re- Urinary Analysis source Center. Here, we used mec2 (dPodocin) RNAi flies To measure proteinuria, urinary albumin levels were mea- (VDRC ID: 104601), Cher (dFilamin-B) RNAi flies (VDRC sured with a mouse albumin ELISA kit (ICL/Dunn Labortech- ID: 107451), and Kirre (dNeph) RNAi flies (VDRC ID: nik GmbH, Asbach, Germany) according to the manufacturer. 27227). To obtain nephrocyte-specificknockdownstrains, In parallel, urinary creatinine levels were measured with a RNAi strains were mated with an Sns (sticks and stones)- urinary creatinine kit (Biomol, Hamburg, Germany). Gal4 driver strain. Flies were kept at 25°C. Doxorubicin Study Nephrocyte Dissection and Immunofluorescence Doxorubicin was injected once into the tail vein at a concentra- Dissection of Drosophila garland nephrocytes was performed tion of 6 mg/kg body wt (for CD-1 background). Doxorubicin with 3rd instar larvae. Isolated nephrocytes were fixed in 4% was dissolved in 0.9% physiologic NaCl, which was also used as formaldehyde for 20 minutes followed by a 1-hour incubation control treatment. Doxorubicin was obtained from the phar- in MeOH at room temperature. Afterward, cells were washed macy of the University Hospital Cologne. The day of injection with staining PBS (PBS15% BSA10.01% Triton-X) three is counted as day 0. At days 7 and 14 urine was collected and used times for 20 minutes. Antibody staining was performed over- for urinary albumin measurement. Animals were euthanized night at 4°C, followed by three washing steps. Antibodies either on day 6 (quantitative PCR) or on day 14 (proteomic were diluted with staining PBS. For antibody dilutions refer analysis) and tissue was either collected for further podocyte to Table 1. Afterward, cells were used for blocking with 5% isolation with FACS or collected for further immunohistochem- NDS in staining PBS for 30 minutes at room temperature. istry. Animals used in this study were 12-week-old male mice. The secondary antibody was diluted 1:250 in staining PBS LPS Study and incubated for 1 hour at room temperature. Subsequently, LPS was injected into the peritoneal cavity once, at a final cells were washed again three times and mounted with Vectashield. concentration of 20 mg/g body wt. LPS was dissolved in Super-resolution images were acquired using a STED microscope 3 0.9% NaCl, and appropriate vehicle treatment was performed (TCS SP8 gSTED 3 , Leica Microsystems) equipped with a white- as control. Urine was collected before the LPS injection and light laser for excitation and sensitive hybrid detectors (HyDs) 3 24 hours after. Animals were euthanized 24 hours after the for time-gated detection. A 100 oil immersion objective with a 3 injection and tissue was either collected for podocyte isolation numeric aperture of 1.4 (PL Apo 100 /1.4 Oil STED, Leica by FACS or harvested for further immunohistochemistry. Microsystems) was used. All images were deconvolved with fi Animals used were from both sexes and were 12 weeks old. Huygens Professional version 17.10 (Scienti c Volume Imaging, The Netherlands, http://svi.nl). Histologic Analysis on Mouse Tissue Mice were euthanized via perfusion with PBS via the heart. Filtration Assay in Drosophila Nephrocytes Kidneys were fixed in 4% paraformaldehyde overnight fol- Nephrocytes of 3rd instar larvae were dissected and incubated lowed by embedding in paraffin. Periodic acid staining was for 1 minute in 0.2 mg/ml FITC-albumin as previously de- performed according to standard methods on 2-mm-thick sec- scribed,15 followed by 1-minute washing and 20-minute fixa- tions. All images were taken with a Leica SCN400 slidescanner tion in 4% formaldehyde. Afterward, the tissue was mounted and further processed using Aperio ImageScope v12.0.1.5030. with Vectashield and imaged with an Axiovert 200M micro- Immunohistochemistry was performed using four different scope and further processed with ImageJ/Fiji 1.50f8 and antibodies, listed in Table 1. After dehydration with increasing Adobe Photoshop Version 11.0. Exposure times were identical alcohol concentrations, sections were placed in a pressure for comparative analyses. To evaluate filtration function, the cooker for antigen retrieval using Tris-EDTA pH 9.0. After mean intensity of FITC was obtained by using ImageJ/Fiji blocking with 3% H2O2 and 1% BSA, the primary antibodies 1.50f8. FITC intensity of SNS/Gal4 control cells was used for (Table 1) were incubated overnight at 4°C. On the next day, the normalization and for statistical analysis. biotin-conjugated secondary antibody was incubated for 1 hour at room temperature, followed by 3,39-diaminobenzidine Sample Preparation for Ultrasensitive Proteomics labeling and hematoxylin counterstain. Podocyte pellets were solubilized with 40 ml8%SDSand heated at 95°C for 5 minutes to solubilize and denature all Histologic Analysis on Human Tissues proteins. Next, 25 mU Benzonase was added to the solution. Embedded biopsy tissue from patients with FSGS was received Microdissected garland nephrocytes from single 3rd instar from the Pathology Department of the University Hospital larvae (five larvae per genotype) were solubilized with 40 ml

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Table 1. Antibodies Name Company Catalog No. Host Species Dilution IF Anti-Duf Provided by B. Denholm, University of Edinburgh, UK N/A Rabbit 1:100 Anti-Pyd Developmental Studies Hybridoma Bank PYD2 Mouse 1:25 Anti-Filamin-B Invitrogen PA5–52098 Rabbit 1:200 Anti-PSMB7 Sigma HPA052408 Rabbit 1:200 Anti-STAT-1 Sigma SAB4300326 Rabbit 1:200 Anti-TRIM32 Proteintech 10326–1-AP Rabbit 1:200 Anti-rabbit-Cy3 Jackson ImmunoResearch 711–165–152 Donkey 1:250 Anti-mouse-Cy5 Jackson ImmunoResearch 715–175–150 Donkey 1:250 Biotin-conjugated anti-rabbit Jackson ImmunoResearch 711–065–152 Donkey 1:400 Abberior STAR 635P Abberior ST635P Rabbit 1:250 Abberior STAR 580 Abberior ST580 Mouse 1:250 IF, immunoﬂuorescence; N/A, not applicable.

8% SDS followed by heating at 95°C for 5 minutes. Human glomeruli. Single glomeruli were harvested as previously de- and rat glomeruli were solubilized in 8% SDS and treated as scribed.17 In brief, glomeruli were isolated by sequential siev- previously described.9 Podocyte and nephrocyte samples were ing and washed using ice-cold PBS. Single glomeruli were reduced with 5 mM DTT at 37°C for 30 minutes. Then, pro- manually picked using 1-ml residual volume. Vehicle-treated teins were alkylated at room temperature with 10 mM IAA in controls were also collected and recorded. Glomeruli were the dark. Proteins were prepared and digested using Trypsin then lysed in SDS and processed using the single-pot, solid- and LysC using the SP3 ultrasensitive proteomics technique as phase sample preparation (SP)3 protocol developed by previously described.9 Hughes et al.,10 including tryptic and LysC digestion. Then, the peptides were run using a parallel reaction monitoring Mass Spectrometry approach in targeted proteomics mode as previously de- Proteomics data acquisition was performed on a quadrupole Or- scribed,9 targeting the peptides listed in Supplemental bitrap hybrid mass spectrometer (QExactive Plus, Thermo) cou- Table 3. Quantiﬁcation of abundance of peptides as well as pled to an easynLC exactly as previously described using 2.5-hour merging of peptide expression into protein abundance was (for podocytes) or 1-hour gradients (for nephrocytes).16 performed using Skyline software18 as previously described.9

Study Approval and Human Patients Bioinformatic Analysis of Proteomics Data All investigations involving human subjects were conducted in Raw files generated by the mass spectrometry were processed accordance with the principles of the Declaration of Helsinki. All using MaxQuant v. 1.5.3.8.19 Briefly, raw data generated by the investigations were conducted after obtaining informed consent mass spectrometer were searched against a database consisting from the patients or their parents. All procedures were approved of a mouse uniprot reference database without isoforms by local ethics committees in Cologne. The described pediatric (downloaded in January of 2017), including common con- patient with a steroid-resistant nephrotic syndrome and biopsy- taminants, or a drosophila uniprot reference database without proven FSGS (7 years old, male) was nephrectomized during isoforms, downloaded in January of 2018. Default search param- living donor transplantation due to massive proteinuria, and eters were used, including mass accuracy of 2.5 ppm, methionine the material was stored on ice, transported to the laboratory, oxidation as a variable modification, cysteine alkylation as a fixed and the kidney was microdissected. Exome sequencing using modification, and 0.01 FDR for peptide spectrum, site, and pro- the Trusight one gene panel was performed and revealed a de- tein identification using the target decoy approach. Match be- scribed TRPC6 variant (heterozygous) and an NPHS2 polymor- tween run options was enabled. The search option also included phism resulting in an R229Q amino acid variant, and no further removal of all peptides that were identified by one peptide and by likely disease-causing variants could be identified using a com- post-translational modification only. The label-free quantitative prehensive exome resequencing approach. The other two pa- (LFQ) algorithm option was enabled to obtain label-free quan- tients, whose material was used for proteomic analysis, were tification intensities.20 Protein expression was further analyzed previously described.9 The patient material used for immuno- using Perseus 1.5.5.3 as well as homemade R-scripts. In brief, histochemistry was obtained from renal biopsy specimens after LFQ expression values were log2 transformed, and common routine diagnostics were performed. Informed consent was ob- contaminants, reverse peptides, and peptides only found by me- tained from all patients. thionine oxidation were removed. Intensity-based absolute quantification (iBAQ), a parameter for absolute protein expres- Single-Glomerular Proteomics sion, was calculated as previously described.21 Data were filtered Rats were treated with PAN (these were the same rats as in a to contain at least two-thirds valid data. Samples including previous study17) and a fraction was used for harvest of single ,50% of total identified proteins were excluded. Imputation

4 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH of missing values was performed (downshift, 1.8 SD; width, 0.3). expressed as means6SEM. Statistical significance was evalu- A two-tailed t test was used, and correction for multiple hy- ated using GraphPad Prism version 6 for Windows (GraphPad pothesis testing was performed using the significance analysis Software, San Diego, CA). For comparison of two groups, t test of microarray approach using a Fudge factor (s0) and a was used. A P value of ,0.05 was considered significant. For permutation-based FDR.22 Parameters were s050.1 and one independent variable, one-way ANOVAcombined with FDR50.05 (200 randomizations) if not otherwise indicated. Tukey’smultiplecomparisontestwasperformed.AP value Hierarchic clustering was performed by Euclidean distance of ,0.05 was considered significant. For two independent (Figure 1) or maximum distance (Figure 2) using k-means variables, two-way ANOVA combined with Sidak’smultiple preprocessing. Gene ontology (GO) term enrichments were comparison test was applied and a P value ,0.05 was performed using a Fisher exact test of increased and decreased considered significant. population versus the nonchanged protein population after annotation with the proteins. For comparison of datasets, log2 ratios of the LPS model were mapped on the log2 ratios RESULTS of the Doxorubicin model by Uniprot identifier. Two- dimensional GO and KEGG enrichment analysis was performed Glomerular Omics Data from Patients with Late-Stage on the log2 ratios of LPS/control versus Doxorubicin/control FSGS Are Biased by the Effect of Podocyte Loss and (FDR threshold 0.05 with 200 randomizations).23 Visualization Dedifferentiation of LFQ intensity ratio on the canonical actin–associated cyto- In order to identify differentially regulated pathways and sig- skeleton KEGG pathway was performed using Pathview with natures in FSGS, we performed proteomics analysis of single the default settings for normalization and visualization glomeruli from two patients with FSGS (Figure 1A). Both (pathview.r-forge.r-project.org).24 Homologene groups patients received bilateral nephrectomy at the time of cadav- (NCBI) were used for annotation of ortholog protein groups eric kidney transplantation due to massive, therapy-resistant between mouse, human, and rat. Z-scoring was performed proteinuria. Nonsclerosed glomeruli were microdissected and using standard statistical procedures. subjected to single-glomerular proteomics analysis as previously described.9 Eighty-five putative podocyte markers, FSGS Signature which were discovered by deep proteomic profiling of mouse The human FSGS signature was created from datasets de- podocytes and validated via human protein atlas staining,28 posited into Nephroseq (www.nephroseq.org). Differential showed a dysregulation of several podocyte-specific proteins expression profiles were precomputed; FSGS versus normal in both patients, including a downregulation of podocyte- kidney analysis was performed on the Hodgin FSGS Glom specific proteases such as DPP4, and an increase in dataset and FSGS versus healthy living donor analysis was podocyte-specific adhesion proteins and cytoskeletal proteins performed on the Ju CKD Glom dataset.25,26 A meta-analysis such as integrins and myoferlin. Extracellular matrix proteins was performed on the two datasets to generate the top such as collagen A6 were increased (Figure 1A). Subsequent 200 over-expressing and top 200 underexpressing genes analysis of these putative podocyte markers in injured glomer- (by median P value across analyses). uli of a patient with nephrin mutation revealed a normally distributed, but overall downshifted, regulation of these pro- Analysis of Mechanical Signature teins, resulting in an underrepresentation of podocyte-specific Protein expression data from mechanically stressed human proteins (Figure 1B). This can also be observed when analyz- podocytes (grown on plastic) and podocytes grown on 12-kPa ing transcriptomic levels in human FSGS tissue (glomeruli) stiff dishes (not stressed) were reanalyzed.4 To find commonly from the studies by Ju and Hodgin combined in Nephroseq regulated proteins, significantly increased proteins in (Figure 1C).25,26 These data suggest that analysis of late-stage Doxorubicin-treated, FACS-sorted isolated podocytes were FSGS tissue might be biased by podocyte dedifferentiation and matched with significantly regulated proteins on the basis of loss, and that an isolated window into early podocyte injury NCBI homologene (https://www.ncbi.nlm.nih.gov/homologene) may be advantageous to identify molecular pathways involved groups. in the podocyte stress response.

Raw Data Proteomics Analysis of Isolated Podocytes after Raw files of the study are deposited under PRIDE/ProteomEx- Doxorubicin Injury change27 under the following identifiers: Project accession: To overcome these limitations, we aimed to generate PXD012064; PXD012063; PXD012025. podocyte-specific proteome datasets and utilized two commonly used FSGS disease models in mice, the Doxorubicin Statistical Analyses and LPS models. Our intent was to investigate proteomic Proteomics analysis, including correction for multiple testing, changes in podocytes as soon as proteinuria was detectably was performed as described under “bioinformatics analysis increased. We induced podocyte injury by intravenous appli- of proteomics data.” For the remaining data, all results are cation of Doxorubicin. After 2 weeks, podocytes were isolated

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Single glomerular proteome: B Single glomerular proteome: A FSGS patients Patient with NPHS1 mutation

4 1.0

(fractions) 0.5 0 relative frequency -2 0.0 -6 -4 -2 0 2 4

FSGS patient 2 -4 log 2 fold change fold change FSGS/control)

2 -6

(log Glomerular transcriptomics -8 C 1.5

-8 -6 -4 -2 0 2 4 6 8 1.0 FSGS patient 1 (log2 fold change FSGS/control)

Podocyte specific gene products (fractions) 0.5 relative frequency

0.0 -2 -1 0 1 2 log 2 fold change

Figure 1. Glomerular omics data from patients with late-stage FSGS are biased by the effect of podocyte loss and dedifferentiation. (A) Comparison of single-glomerular proteomes from two patients with late-stage FSGS. The fold change of the regulation versus control is plotted. Podocyte-specific proteins are in blue and are either strongly up- or downregulated. (B) Protein distribution of single-glomerular proteomics from one patient with NPHS1 mutation presented as a cumulative histogram. This revealed an underrepresentation of podocyte-specificproteinsincomparisonwiththewholeglomerularproteome.Podocyte- specific proteins, n585; other proteins, n51079; Kolmogorov–Smirnov test: P,0.001. (C) Transcript distribution of glomerular transcriptomics revealed an underrepresentation of podocyte-specificproteinsincomparisonwiththewholeglomerulartran- scriptome. Podocyte-specific gene transcripts (corresponding to podocyte-specific proteins), n5308; other proteins, n514,434; Kolmogorov–Smirnov test: P,0.001. by FACS and subjected to proteomic analysis per individual Table 4). We injected LPS intraperitoneally into mice and an- podocyte mouse fraction using an ultrasensitive proteomics alyzed protein expression in FACS-sorted podocytes. LPS ad- workflow9,10 (Figure 2, A–C). The experimental mice showed ministration resulted in slight proteinuria (Figure 3B) and considerable variability in the degree of disease phenotype, only minor morphologic changes (Figure 3C). Differential most likely due to the early timepoint of termination. Ultra- proteomic data analysis revealed that 120 proteins were sig- sensitive proteomic analysis identified 2200 proteins, of which nificantly changed upon podocyte injury. Although a similar 225 were significantly up- or downregulated (Figure 2, D and increase of Stat-1 was observed in both models of podocyte E). Among the increased proteins were several known glomer- injury (Figure 3, D and E), overall, there was only a weak ular disease–associated proteins, such as the transcription fac- correlation of damage-regulated protein expression between tors Stat-1 and Stat-329,30 (Figure 2E, Supplemental Table 1). the two models (R50.22;Figure3F).Inaddition,weper- Protein copy number estimations using the iBAQ parameter21 formed two-dimensional enrichment analysis using GO showed that the dynamic range of the podocyte proteome biologic processes (Figure 3G) and KEGG pathways comprised five orders of magnitude (Supplemental (Supplemental Figure 2A). Both databases revealed Figure 1). GO enrichment revealed an upregulation of meta- damage-induced upregulation of proteins in essential met- bolic processes, whereas adhesion processes were downregu- abolic pathways such as fatty acid metabolism, glycolysis, lated (Figure 2F). oxidative phosphorylation, and acetyl-CoA metabolic and catabolic processes. In addition, both models showed a sig- Proteomics Analysis of Pure Podocyte Injury in LPS nificant downregulation of processes and pathways associ- Damage ated with the actin-cytoskeleton, such as focal adhesions Next, we asked whether similar proteins would be changed in (Supplemental Figure 2A) and actin-sensitive pathways the LPS model of podocyte damage (Figure 3A, Supplemental (Supplemental Figure 2B).

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AB Doxorubicin Saline Saline Doxorubicin

Albuminuria n=5 n=5

Isolation of primary podocytes

Doxorubicin day 14

105

104 Podocytes 6.5 PE-A 0 C

6.0

0102 103 104 105 Saline treated 5.5 FITC-A Doxorubicin treated 5.0 ultrasensitive sample 1.5 preparation (SP3) 1.0

nLC-MS/MS 0.5 Albumin/ Crea ratio [mg/mg]

0.0 day 0 day 7 day 14 D E

5.5 LFQ 5 intensity 20 26 32 4.5 4

Doxo Doxo Doxo Doxo Doxo 3.5 Albumin/ Crea 6 control control control control control ratio [mg/mg] 2 3

- log p 2.5 2 1.5 1 0.5 0

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5

LFQ log2 (Doxorubicin/ control) F Decreased GO terms Increased GO terms protein mitochondrion polymerization cofactor metabolic process cell activation oxidoreductase activity ion transport cell adhesion cellular lipid metabolic process biological lipid metabolic process adhesion intracellular membrane- bounded organelle 01234 01234 fold change fold change

Figure 2. Proteomics analysis of isolated podocytes damaged by Doxorubicin revealed an upregulation of metabolic process, while adhesion processes were downregulated. (A) Workﬂow of Doxorubicin administration and sample preparation. NPHS2.Cre x mT/mG mice were treated with Doxorubicin (6 mg/kg body wt) or saline solution. FACS sorting of GFP-positive podocytes was performed. FACS-sorted podocytes were subjected to proteomic analysis by mass spectrometry. PE-A: tomato; FITC-A; GFP; red dots, Tomato- positive cells; green dots, GFP-positive cells; purple dots, cells out of gating. (B) Periodic acid-Schiff staining reveals morphologic changes upon Doxorubicin treatment in mice. Scale bar, 100 mm. (C) Albumin-to-creatinine ratios of animal urine after 7 and 14 days of Doxorubicin treatment. (D) Proteomic analysis of isolated podocyte fractions. Heatmap depicting LFQ protein expression between control and Doxorubicin-treated animals. Proteinuria per animal is depicted as a bar graph. (E) Volcano plot shows differentially

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Conserved and Individual Features of Podocyte (Supplemental Figure 3, A–C). We also used immunohisto- Damage chemistry to analyze Filamin-B expression in glomeruli of Next, we investigated whether podocyte-specific proteins were 3–4-week-old podocyte-specific Podocin knockout mice primarily affected by podocyte damage across both models. To (Supplemental Figure 3, B–D), a genetic model of FSGS. We this end, we aligned the fold change expression in the two identified injured glomeruli with an increased Filamin-B sig- models with quantitative proteomics data that profiled the nal in podocytes. In parallel, we assessed the expression levels podocyte proteome at a depth of .9000 proteins and com- of the three other candidates, STAT-1, TRIM32, and PSMB7 pared it with nonpodocyte glomerular cells.28 Both podocyte- (Supplemental Figure 3D). In line with data obtained from specific and podocyte-depleted proteins were almost equally the human FSGS samples, we observed an increase of all three affected by each injury stimulus, and both models differed proteins in injured glomeruli, suggesting that these proteins strongly regarding individual protein regulation (Figure 4A). could be part of a stress responses in the podocytes. In our dataset, the majority of the detected proteins were in fact podocyte enriched (Figure 4B). Filamin-B Abundance Increases with Proteinuria in We compared the changed proteins with the initial data- Animals and Single Glomeruli and Is Altered by sets from patients with glomerular disease (Figure 4C). Only Mechanical Stress ahandfulofproteinsweresignificantly upregulated in the Intrigued by the upregulation of the mechanosensor Filamin- human tissue and in the injured podocytes by Doxorubicin B in both human glomeruli and podocytes from Doxorubicin- (Figure4D)andbyLPS(Figure4E).Amongthesewere treated mice, we looked at the correlation of expression levels Filamin-B, Stat-1, and ATP1B1. Filamin-B was also in- with the amount of proteinuria in individual mice treated with creased in transcriptomic datasets of FSGS glomeruli as Doxorubicin. This resulted in a high positive correlation compared with controls (fold change51.27, FDR Q value (R50.76, top 5% ranked percentile of correlation among all ,0.001, obtained from Nephroseq datasets25,26). ATP1B1, proteins in the dataset) and emphasized the potential role of a subunit of the sodium potassium ATPase, was not regu- Filamin-B during podocyte injury (Figure 5A). In addition, we lated in transcriptomic datasets, despite proven expression investigated whether Filamin-B mRNA levels were altered by in podocytes.31 Doxorubicin-induced podocyte injury by performing quanti- Next, we analyzed whether stress response proteins iden- tative PCR on glomeruli isolated from mice either without tified in these isolated podocyte proteomics experiments Doxorubicin treatment (baseline) or with Doxorubicin treat- could have a broader relevance for podocyte injury. As a ment (Figure 5B). This analysis revealed a significant upregu- proof of concept, we decided to analyze the role of lation of Filamin-B mRNA levels at day 6 after Doxorubicin Filamin-B, a mechanical stress response protein, in different administration. conditions of glomerular disease. We performed immunos- To further substantiate the functional role of Filamin-B in taining of human biopsy samples from five patients with podocyte injury, we analyzed its amount in single glomeruli FSGS as well as controls from kidney biopsy samples, reveal- from the PAN proteinuria model in rats using targeted pro- ing strong expression of Filamin-B in certain, but not all, teomics (Figure 5C). Glomerular-accumulated albumin is patients with FSGS. We did observe that Filamin-B staining consistently found and quantified in glomerular proteomics intensity appeared to be concurrent with the degree of pro- studies.32 A single-glomerular proteomics approach allows to teinuria at the time of biopsy (proteinuria parameters for detect markers of glomerular injury such as albumin and to biopsies of the depicted patients were: patient 1: 7676 mg/g correlate them with the expression of individual proteins.9 albumin-to-creatinine; patient 2: 1715 mg/g albumin-to- When individual glomerular proteomes were resolved, creatinine; patient 3: 6333 mg/g albumin-to-creatinine). Filamin-B correlated significantly with the amount of albumin Representative results of the patients are presented in detected in every glomerulus (Figure 5D). In line with our Supplemental Figure 3, A–C. For further corroboration, in vivo data, reanalysis of published in vitro data, podocytes we also selected three additional candidates on the basis of mechanically stressed by cultivation on plastic dishes as their significant upregulation in podocytes isolated from opposed to physiological stiffness,4 also revealed an increase Doxorubicin-treated mice. These candidates were the tran- of Filamin-B protein upon elevated mechanical stress in scription factor Stat-1, the E3 ubiquitin ligase TRIM32, and podocytes, as part of a group of proteins that was also the proteasome component PSMB7. All of these candidates increased in the Doxorubicin-treated podocytes (Figure 5E).4 were visualized on human kidney tissue from patients with Taken together, our data obtained from human, rat, and FSGS and revealed an upregulation of the respective pro- mouse tissues as well as published in vitro data suggest a role of tein in podocytes in some, but not all, of the patients Filamin-B in the podocyte stress response.

regulated proteins when comparing proteinuric Doxorubicin versus control animals. Prioritized candidates such as Flnb (Filamin-B), STAT-1, PSMB7, and TRIM32 are depicted in red. (F) GO term enrichment analysis revealed an upregulation of metabolic processes, whereas actin-cytoskeleton–associated proteins were downregulated.

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A LPS Saline B C Saline

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Figure 3. Proteomics analysis of isolated podocytes injured by LPS revealed a downregulation of pathways associated with the actin- cytoskeleton. (A) Workﬂow of LPS administration and sample preparation. Podocin.2A.iCre.2A.mTomato mice were treated with LPS (20 mg/g body wt) or saline solution. FACS sorting of Tomato-positive podocytes was performed. FACS-sorted podocytes were subjected to proteomic analysis by mass spectrometry. (B) Albumin-to-creatinine ratios show a signiﬁcant increase in urinary albumin 24 hours after LPS

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Filamin-B/Cher Modulates Nephrocyte Function without that can be directly linked to phenotypes of complex tissues.37 Affecting Nephrocyte Slit Diaphragm Structure The advantage of the proteome as a rather close correlate to Filamin-B could therefore contribute to the podocyte re- the phenotype is, however, largely limited by its practicability sponse to injury. To investigate this hypothesis further and because it requires large preparations of renal cells from model to identify mechanisms influenced by altered Filamin-B levels, organisms. Here, we expanded proteomic acquisition to a we employed the model organism Drosophila and its nephro- multitude of experimental systems to investigate model or- cytes. We generated flies with a nephrocyte-specificdepletion ganisms of podocyte injury to improve the accessibility of of Cher, the Filamin homolog in the fly, and analyzed neph- proteomics(anoverviewofthestudiesisprovidedin rocyte morphology and function. Supplemental Figure 4). This allowed us for the first time in To identify downstream targets and signaling pathways reg- a proof-of-principle experiment to detect patterns of several ulated by Cher, we performed ultrasensitive proteome analysis of proteins in multiple models in a scalable approach and to Cher-depleted garland nephrocytes from single larvae (3rd in- correlate the phenotype and the extremely variable degree of star), comprising approximately 20 nephrocytes (Figure 6, A and proteinuria with omics perturbations. B, Supplemental Table 2). The proteome was profiled at a depth Podocyte injury causes a stress response followed by podocyte of 1400 proteins over five orders of magnitude and contained detachment and depletion, resulting in an underrepresentation several proteins associated with podocyte disease phenotypes, of podocyte-specificproteins—a problem that involves several among these dMec2/Podocin and dActinin (Figure 6A, aspects contributing to a “bulk” glomerular omics datapoint: Supplemental Table 2).33,34 The proteome data also revealed a alterations of cell fractions in a glomerulus by podocyte loss, significant reduction of Cher expression in knockdown nephro- increase of mesangial and extracellular matrix, reactions of pa- cytes, proving the efficiency of the RNAi strain used (Figure 6B). rietal epithelial cells (PECs), alterations in serum composition, In addition, we identified actin-cytoskeleton–associated and alterations and depositions of complement and serum proteins, matrix-associated proteins to be upregulated upon loss of and many more. Although podocyte responses have been sug- Cher. Among those are Syb-RA (Synaptobrevin), the Vamp2 gested as the key driver in FSGS, it appears to be difficult to ortholog; Zip, the Myh9/10 homolog; Shi, the Dynamin homo- extrapolate a podocyte response from late-stage FSGS samples, log; and Tnc, a matrix-associated protein (Figure 6B). especially because podocyte-specific proteins are underrepre- To assess filtration function, we performed FITC-albumin sented (Figure 1). Here, we therefore aimed to observe the early uptake assays (Figure 6C), which test albumin uptake in neph- fate determining factors of podocyte injury by performing rocytes. Loss of two nephrocyte-specific proteins, Duf (dNEPH) podocyte proteomics from isolated podocyte fractions for and Mec2 (dPodocin), revealed a significant decrease in the the first time. FITC-albumin uptake capacity as previously described.15 To gain an understanding in early models of podocyte in- Knockdown of Cher, in contrast, resulted in a significant injury, we profiled isolated podocytes from two distinct models crease of FITC-albumin uptake. of podocyte injury in mice. These are two widely used models In order to observe whether these alterations were associated of chemical podocyte injury, and the experiment was timed to with a change in the structure of the nephrocyte diaphragm, a catch immediate reactions at the first significant presence of structure similar to the mammalian slit diaphragm,35,36 we also proteinuria. From a mechanistic standpoint, the two models analyzed nephrocyte ultrastructure by super-resolution micros- do not appear to have too much in common. Only computa- copy (Figure 6D). These data showed that the nephrocyte di- tional pathway analysis identified perturbed pathways, which aphragm structure was disturbed upon loss of Mec2, and more were not evident on the individual protein levels itself dramatically changed when Duf was depleted in nephrocytes. (Figure 3G). One of the few proteins found to be increased However, loss of Cher, the Filamin homolog, did not result in a in both disease models was Stat-1, which is active in early changed ultrastructure of the nephrocyte diaphragm. stages of diabetic nephropathy in glomerular compartments in patients,29 and is also increased in kidney tissue of patients with FSGS,38 as confirmed also in this study (Supplemental DISCUSSION Figure 3, A–C). The JAK/STAT pathway plays a crucial role in different processes such as cell division, cell death, and immu- A still unresolved challenge of any transcriptomic analysis is nity. Moreover, JAK2 was described to be important in facil- the insufficient prediction of protein expression abundance itating autophagy in podocytes.39 Notably, STAT activity was administration. One-way ANOVA: *P,0.05. (C) Periodic acid staining reveals no severe morphologic changes in LPS-treated animals. Scale bar, 100 mm. (D) Heatmap representing the clustering of control and LPS-treated mice. (E) Volcano plot depicting differentially regulated proteins in FACS-sorted native podocytes after administration of LPS (24 hours). Candidates with direct physiologic links to podocyte disease are marked, including: Stat1, Stat3, and Nphs1. (F) Scatter plot analysis of the LFQ alterations of Doxorubicin- and LPS-treated animals reveals a common upregulation of Stat1; however, a common global regulation trend between the two models could not be observed. (G) GO term two-dimensional enrichment comparing Doxorubicin- and LPS-treated animals using the GO biologic processes dataset revealed an upregulation of metabolic signaling and a decrease in actin-cytoskeleton–associated pathways upon podocyte injury in both models.

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A B C

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Figure 4. Comparison of murine and human proteomic data sets identified conserved and individual features of podocyte damage. (A and B) Comparison of a wild-type mouse podocyte proteome with the Doxorubicin and the LPS datasets reveals that a majority of the detected proteins are podocyte specific and some of them are decreased upon injury. (C) Comparison of homologous protein expression in Doxorubicin- and LPS-treated FACS-sorted native podocytes with single human glomerular FSGS samples revealed only minor similarities. (D) Individual plotting and statistical outlier testing (FDR,0.01) revealed two interesting candidates, Filamin-B and ATP1B1. Both are significantly upregulated in the Doxorubicin model and the human FSGS sample. (E) Plotting of the LPS samples versus the human FSGS samples did not reveal increased statistical outliers in either condition. increased in the entire kidney, consistent with the cited pre- we know from previous studies that E3 ubiquitin ligases play vious reports.38 Of note, other models such as APOL1 and an important role in podocyte biology,16,43 nothing is known suPAR transgenic mice might be modeling human pathophys- about the functional role of TRIM32 in podocytes. TRIM32 is iology more closely.40–42 a nuclear-localized E3 ligase that has been shown to regulate Two other interesting proteins, which were both upregu- the antiviral response in neurons, in particular HIV related upon Doxorubicin-induced injury and in human FSGS sponse.44,45 PSMB7 is part of the 20S proteasome complex. samples, are TRIM32 and PSMB7 (Figure 1, Supplemental Both of these are part of proteostatic responses in the kidney.46 Figure 3). TRIM32 functions as E3 ubiquitin protein ligase. The high-resolution approach provided here potentially One of its substrates is Dysbindin, which plays a role in actin- allows the mathematic correlation of phenotypic parameters cytoskeleton reorganization and neurite outgrowth. Although (proteinuria) and protein abundance. This approach can be

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Saline treated A B 15 Doxorubicin treated * R=0.76 -0.5 10 -1 -1.5 delta CT

-2 2 5

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log -3 -3.5 0 -2 -1.5-1 -0.5 0 0.5 Baseline Doxorubicin Day 6 log2 urinary Albumin

C PAN treated rat, D PAN treated rat, E 12 single glomeruli single glomeruli Gsr Rps11 Zranb2 Alb Xpo5 1: in vivo 10 Rps9 log2 (Doxo/control) 4 Idh2 Hspe1 2: in vitro Flnb 8 3 log2 (Plastic/12k PA) Trap1 2 Ldhb (Albumin) Rrbp1 2 6 1 Psma7 log Plod2 0 Cald1

-log (pvalue) 4 Hadha -2 -1 0 1 Hadhb Aco2 Cd2ap log2 (Filamin B) Hspa9 2 Nphs1 Lamp2 Phb2 Nphs2 Animal 1 Psmb1 Animal 2 Ogdh log2 fold change 0 Animal 3 Ctnna1 -2 +2 Sdha 024

log2 LFQ (PAN/control)

Figure 5. Filamin-B abundance increases with proteinuria in animals and single glomeruli and is altered by mechanical stress. (A) Correlation analysis revealed a strong positive correlation of podocyte Filamin-B protein with urinary albumin concentration (R, Pearson’s correlation coefficient; P,0.05). (B) Quantitative PCR of glomeruli isolated from Doxorubicin-treated mice revealed a sig- D nificant increase of Filamin-B mRNA levels (2 CT) upon injury. Filamin-B levels are normalized to HPRT. t test: *P,0.05. (C) Volcano plot of PAN-treated single rat glomeruli reveals a significant increase of albumin (Alb), whereas podocyte-specific proteins such as Nphs2 and Nphs1 are downregulated. (D) Correlation analysis of Filamin-B levels with albumin reveals a positive correlation, R50.76. (E) Comparison of protein abundance in our Doxorubicin-treated podocytes with cell culture podocytes exposed to increased mechanical stress (when grown on plastic as opposed to 12-kPa soft matrices) revealed similar signatures with regard to mechanosensation. used to find physiologic regulatory circuits and associations. high expression of Filamins, a ubiquitously expressed protein As a proof of concept, we investigated the functional role of family, is strongly associated with mechanical stress condi- Filamin-B in greater detail. We discovered that the protein tions in other tissues and cells, including podocytes.49–52 abundance of the mechanosensor protein Filamin-B corre- Examination of individual glomeruli from the PAN model lated positively with elevated proteinuria in individual mice in rats revealed coregulation of Filamin-B with deposited al- (Figure 5). In line with this, tissue from human patients with bumin in individual glomeruli proteomes from proteinuric FSGS also showed increased Filamin-B on protein and tran- rats. Albumin is frequently found in glomerular tissue from script levels. Filamin-B is a cytoskeleton-associated protein proteinuric rat models, and the amount reflects the proteinu- that has been described as part of a mechanosensory complex ric degree of the individual animal.4,37 In addition, we utilized at the slit diaphragm,47 and that is compensatorily upregula- the model organism Drosophila and its nephrocytes and as- ted via a selective autophagy–dependent mechanotransduc- sessed effects after Cher (dFilamin) depletion. This revealed an tive feedback loop involving YAP.48 It should be noted that increased filtration upon loss of Cher, whereas the nephrocyte

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A Single garland proteome (“control-Gal4“) B 3.5 8 Zhang et el. 2013 strong phenotype Zhang et al. 2013 7 Gdi weak phenotype 3 Rab1 Fu et al.2017 porin ATPsyn-b RhoGDI Chc 6 Mlc1 Arf102F CG10688 Sar1 Cubn Rac1 Actn Ca-P60A 5 REG RhoGAP68F 2.5 Clc Mec2 Rab8 log10(iBAQ) rhea Cdc42 Myo61F LanB1 4 Sply 2 3 400 800 1200

Rank IBAQ -log p 1.5 C p<0.05 2.5 1 **** 2.0

0.5 1.5 ****

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0.5 -4 -3 -2 -1 0 1 2 3 log (Cher knockdown/control-Gal4) mean FITC-Albumin intensity 2 0.0 Control Cher kd Duf kd Mec2 kd

D Control (Sns-Gal4/w) Control (Sns-Gal4/w) magnification Cher kd

Duf kd Mec2 kd Mec2 kd magnification Duf / Pyd

Figure 6. Filamin-B/Cher modulates nephrocyte function and is not required for nephrocyte slit diaphragm structure. (A) Dynamic range of the nephrocyte proteome from a single fly covers 4.5 orders of magnitude. iBAQ is plotted. Proteins with a known phenotype from Zhang et al. and Fu et al. are marked.33,34 (B) Volcano plot depicting differentially regulated nephrocyte proteins upon Cher loss. Downregulated proteins such as Cher are depicted in red, whereas upregulated proteins such Syb-RA and tnc are shown in blue. n54 flies per group. (C) Loss of Cher (dFilamin) causes a significantly increased FITC-albumin uptake when compared with control flies. As positive control, we used a Duf (dNeph) and a Mec2 (dPodocin) knockdown strain. n53 (three independent matings and 5–7animals per mating; total number of observations per group: Control: 244, Cher kd: 73, Duf kd: 58 and Mec2 kd: 108); ****P,0.001. (D) Nephrocyte-specific depletion of Cher (dFilamin) does not cause morphologic changes as depicted for Duf (dNeph) and Pyd (dZO-1) localization, when compared with control flies (Sns-Gal4/w). As positive control, Duf and Mec2 knockdown nephrocytes were imaged. Loss of Duf presented with a severe morphologic phenotype and an almost complete loss of the nephrocyte diaphragm. Scale bar, 5 mm; identical for all panels.

JASN 31: ccc–ccc,2020 Native Podocyte Injury Proteomics 13 BASIC RESEARCH www.jasn.org diaphragm morphology was maintained. Although this model supported by a German Research Foundation fellowship (RI2811/1-1). This can be viewed as rather distant from human biology, the Dro- project was part of the work performed in FOR2743 to Dr. Höhfeld (HO1518/ 13-1), Dr. Rinschen (RI2811/2-1), and Dr. Benzing (BE2212/25-1). The Anti- sophila nephrocyte is still one of the fastest models to analyze 54 fi Pyd mAb developed by Fanning was obtained from the Developmental Stud- ltration function and nephrocyte diaphragm integrity and is ies Hybridoma Bank, created by the National Institute of Child Health and used to model podocyte function.15,35,53 Human Development of the National Institutes of Health, and maintained at Taken together, the combined data suggest that protective The University of Iowa, Department of Biology, Iowa City, IA. Dr. Koehler mechanisms against mechanical forces play an important role received funding from the German Research Foundation (KO 6045/1-1), the in mechanotransduction and modulation of podocyte func- Else-Kröner-Fresenius Stiftung (2017_A135), and the Köln Fortune Program of the University of Cologne, Germany. tion and morphology. In addition, we advanced proteomics from the analysis of pooled glomerular samples from several mice to podocyte fractions from an individual mouse. Com- SUPPLEMENTAL MATERIAL bined with single-glomerular data from human nephrectomies and animal models, this allows identification of functional and phenotype-associated protein expression patterns that govern This article contains the following supplemental material online at the podocyte stress response. http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019030312/-/ DCSupplemental. Supplemental Figure 1. Dynamic range. Protein copy numbers of podocytes isolated from a single mouse. ACKNOWLEDGMENTS Supplemental Figure 2. Different modes of injury cause an upregulation of metabolic signaling and a downregulation of actin- cytoskeleton–associated pathways. We acknowledge the patients and their parents who donated bio- Supplemental Figure 3. Immunohistochemistry on human FSGS material for this study. We thank V. Ludwig, R. Herzog, G. Rappl, the and Podocin knockout tissue revealed an increased Filamin-B Cologne Excellence Cluster on Cellular Stress Responses in Aging- expression. Associated Diseases (CECAD) imaging facility, and the CECAD Supplemental Figure 4. Overview of this study, demonstrating proteomics facility for excellent technical support. the applicability of sensitive proteomics for phenotype-proteome Dr.Koehler,Dr.Rinschen,andDr.Brinkkoetterconceivedthe correlations. study; Dr. Koehler, Dr. Kuczkowski, Dr. Kuehne, Dr. Jüngst, and Supplemental Table 1. Proteome data from the doxorubicin study. Dr. Rinschen performed experiments; Dr. Koehler and Dr. Rinschen Supplemental Table 2. Nephrocyte proteome data. analyzed the data; Dr. Hoehne, Dr. Grahammer, Dr. Eddy, Dr. Kretzler, Supplemental Table 3. List of peptides used in targeted proteomics. and Dr. Beck contributed new tools, reagents, animal models, or bio- Supplemental Table 4. Proteome data from the LPS study. material; Dr. Koehler and Dr. Rinschen made the figures and drafted the paper; Dr. Koehler, Dr. Rinschen, Dr. Brinkkoetter, Dr. Schermer, Dr. Benzing, and Dr. Höhfeld revised the paper; all authors approved REFERENCES the final version of the manuscript.

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AFFILIATIONS

1Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany; 2Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom; 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; 4III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Eppendorf, Hamburg, Germany; 5Division of Nephrology, Department of Internal Medicine, and 6Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; 7Department of Human Genetics, University Hospital Cologne, Cologne, Germany; 8Cell Biology, University of Bonn, Bonn, Germany; and 9Center for Metabolomics and Mass Spectrometry, The Scripps Research Institute, La Jolla, California

16 JASN JASN 31: ccc–ccc,2020